As highlighted in the 'Bad Bugs, No Drugs' campaign by the Infectious Diseases Society of America (IDSA), There simply aren't enough new drugs in the pharmaceutical pipeline to keep pace with drug-resistant bacterial infections, so-called 'superbugs'. Numerous hospitals worldwide have experienced outbreaks of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter species, Enterococcus faecium and Staphyloccocus aureus. All of these pathogens are on the IDSA 'hit list' of the six top-priority dangerous microorganisms ESKAPE that require the most urgent attention to discover new antibiotics. Sadly, no novel antibiotics against MDR P. aeruginosa, A. baumannii and K. pneumoniae will be available for many years to come. Polymyxins (i.e. colistin and polymyxin B) are now being used as the 'last-line' of therapy for infections caused by these very problematic MDR pathogens. Very unfortunately, emergence of polymyxin resistance has been increasingly reported recently. In essence, resistance to polymyxins implies a total lack of antibiotics for treatment of life-threatening infections caused by these Gram-negative bacteria. Research Design: Using a new structure-activity relationship (SAR)-based mechanistic model for polymyxins, novel lipopeptides we designed and synthesized showed very promising activity against polymyxin-resistant MDR strains of P. aeruginosa, A. baumannii and K. pneumoniae, while maintaining activity against polymyxin- susceptible strains. Remarkably, a number of these lipopeptides were also active against MDR strains of two Gram-positive 'superbugs'. Our approach, based upon modeling the interaction between polymyxins and the lipid A of lipopolysaccharides (LPS), is novel. Our hypothesis is that polymyxin resistance in Gram-negative and/or Gram-positive bacteria is overcome by modifications of the core polymyxin structure. A systematic funneling approach with feedback loops will allow us to 'learn' as much as possible at early stages about the SAR to inform the design of superior lipopeptides. Aim 1: To design, synthesize and evaluate microbiologically ~125 novel lipopeptides active against: (a) polymyxin-resistant Gram-negative 'superbugs' using an SAR-based mechanistic model; and (b) problematic MDR Gram-positive E. faecium and S. aureus. Three series of lipopeptides will be designed and synthesized to enhance (i) hydrophobic interactions, (ii) polar interactions, and (iii) a combination of both, with lipid A. Pharmacological evaluations in Specifi Aims 3 and 4 will provide important information for improvement of the model and design of superior lipopeptides. Aim 2: To elucidate the mechanism(s) of activity of the lipopeptides against both Gram-negative and -positive pathogens. Confocal microscopic, transcriptomic and biochemical approaches will be employed to examine the antibacterial activity of our lipopeptides against both Gram-negative and -positive pathogens. The results will provide valuable mechanistic information for designing more active lead lipopeptides (Specific Aim 1). Aim 3: To conduct lead optimization based upon characterization of pharmacodynamics, potential for development of resistance and toxicity, and physicochemical and pharmacokinetic properties. In the lead optimization process, we will measure the minimum inhibitory and bactericidal concentrations of lipopeptides against a large panel of strains. Potential for development of resistance, hemolysis, cytotoxicity and nephrotoxicity, and physicochemical and pharmacokinetic properties, will be assessed. In addition, the interactions with LPS will be studied. Information obtained will be fed back to improve the SAR model (Specific Aim 1). Advanced lead lipopeptides will be identified and proceed to animal studies in Specific Aim 4. Aim 4: To evaluate the in vivo efficacy, toxicity and PK/PD properties for 5 - 10 superior lipopeptides using animal infection models, and to identify 1 - 2 candidates for IND/Phase-1 evaluations. Neutropenic mouse thigh and lung infection models will be employed to evaluate the in vivo efficacy of the advanced leads. Formulations for intravenous delivery will be developed for the candidates before the first dose in man. Aim 5: To conduct IND-enabling toxicology studies. Scale-up of the active pharmaceutical ingredient to conduct range-finding toxicology studies in rats and non-rodent species, and ultimately IND-enabling GLP toxicology and safety pharmacology studies. Even though it is beyond the scope of this RFA, we are very enthusiastic that the identified candidates will be taken to the IND/Phase 1 study with financial support from Mpex. Significance: As highlighted in the WHO World Health Day 2011, no action today means no cure tomorrow. This project holds great promise for development of novel antibiotics against the six top-priority ESKAPE 'superbugs' identified by the IDSA, in particular polymyxin-resistant MDR P. aeruginosa, A. baumannii and K. pneumoniae. Overall, this project targets the urgent global unmet medical need and responds in a timely manner to the recent global call for discovery of new antibiotics: The 10 x '20 Initiative.